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Interleukin-23 (IL-23) responsive group 3 innate lymphoid cells (ILC3s) have been implicated in immune homeostasis and pathogenesis in the adult, but little is known about their roles in the newborn. Here we show that IL-23 promotes conversion of embryonic intestinal Lin(-)IL-23R(+)Thy1(+) cells into IL-22-producing Thy1(+)Sca-1(hi) ILC3s in vitro. Gut-specific expression of IL-23 also activated and expanded Thy1(+)Sca-1(hi) ILC3s, which produced IL-22, IL-17, interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) and were distinct from canonical CD4(+) lymphoid tissue inducer (LTi) cells. These ILC3s accumulated under the epithelium in intercellular adhesion molecule (ICAM)-1-positive cell aggregates together with neutrophils that disrupted the epithelium, leading to the formation of discrete intestinal erosions, bleeding, and neonatal death. Genetic and antibody depletion of ILC3s rescued the mice from neonatal death. Antibiotic treatment of pregnant mothers and offspring prolonged survival of IL-23 transgenic mice, suggesting a role for the commensal flora on ILC3-induced pathogenesis. Our results reveal a novel role for the IL-23-ILC3s axis in the pathogenesis of neonatal intestinal inflammation.

Cigarette smoke (CS)-induced lung injury involves innate immune responses. The activation of innate effector cells is thought to require cross talk with dendritic cells (DCs) and macrophages, but the mediators of interaction are unknown. One candidate, CC chemokine receptor 4 (CCR4), is expressed by innate and adaptive effector cells, and its ligands are produced by DCs and macrophages. Using flow cytometry and confocal microscopy, we defined innate responses of lung myeloid DCs, macrophages, and conventional natural killer (NK) cells in mice exposed to CS over 4 days and examined the contribution of CCR4 using CCR4 knockout (CCR4(-/-)) mice. CS affected populations differently, causing an increase in F4/80(+) macrophages, a reduction in parenchymal CD11c(+)CD11b(+)CD103(-) DCs, but no effect on mucosal CD11c(+)CD11b(-)CD103(+) DCs. CS also induced a population of primed/activated CD69(+) NK cells and bronchoepithelial expression of the stress-related NKG2D receptor-activating protein, retinoic acid early transcript 1. CS-exposed CCR4(-/-) mice were similar to controls regarding effects on DCs and macrophages but displayed substantially impaired NK priming/activation and reduced expression of transcripts for interferon gamma, CXCL10, and retinoic acid early transcript 1. Quantitative confocal microscopy revealed that lungs of CS-exposed CCR4(-/-) mice had significantly reduced contacts of NK cells with CD11c(+) cells. These findings demonstrate that acute CS exposure elicits NK cell responses and suggest that CCR4 promotes NK cell priming/activation by mediating contacts with sentinel cells in the lung., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Cysteine-cysteinyl chemokine receptor 4 (CCR4) is expressed by a variety of T-cell subsets and leukocytes. This study examined the participation of CCR4 in response to pulmonary infection with Mycobacterium bovis Bacille-Calmette-Guerin (BCG). Constitutive and induced CCR4 agonist expression was detected among large mononuclear cells. The course of infection and mobilization of effector cell populations were then analyzed in CCR4 knockout (CCR4(-/-)) mice. Compared with controls, CCR4(-/-) mice displayed delayed innate stage (<2 weeks) bacterial clearance and reduced late stage inflammation. Innate impairment was associated with reduced natural killer cell activation. In the adaptive phase, CCR4(-/-) mice generated effector T cells in draining lymph nodes and accumulated effector T cells in lungs, which resulted in normal adaptive stage bacterial elimination at 2 to 4 weeks. However, during the late stage, CCR4(-/-) mice had reduced interferonγ+CD4(+)α/β+ (Th1) and interleukin (IL)-17+CD4(+)α/β+ (Th17) T helper cells in lungs. In contrast, IL-17+ γ/δ T cells in lungs were unaffected. When challenged with mycobacterial antigen- (Ag-) Ag-coated beads to elicit a recall granulomatous response, CCR4(-/-) mice displayed abrogated recall granuloma formation and reduced interferon γ+ Th1 cells. These findings indicate that CCR4 supports innate natural killer cell activation and sustains later CD4(+) Th effector/memory antimycobacterial responses in the lung but is redundant in the early adaptive elimination phase., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

To investigate the role of CD11c(+) cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 microg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F(2alpha), a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c(+) cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge.

Acute graft-versus-host disease (GVHD) and leukemic relapse are serious complications of allogeneic stem-cell transplantation (SCT). Recruitment of activated T cells to host target tissues or sites of leukemic infiltration (graft-versus-leukemia [GVL]) is likely mediated by chemokine receptor-ligand interactions. We examined the contribution of donor cell CCR1 expression to the development of GVHD and GVL using a well-established murine SCT model (B6 --> B6D2F1) and CCR1-deficient mice (CCR1(-/-)). Allo-SCT with CCR1(-/-) donor cells significantly reduced systemic and target organ GVHD severity, and CCR1 expression on both T cells and accessory cells contributed to GVHD mortality. Significant GVL activity was preserved following CCR1(-/-) SCT, but the survival advantage diminished with increasing tumor burden. We then explored the effects of CCR1 expression on allo-specific T-cell responses. Although cytolytic effector function was maintained on a per-cell basis, T-cell proliferation and IFNgamma secretion were significantly reduced both in vivo and in vitro. T-cell function was partially dependent on interactions between CCR1 and CCL5. Collectively, these data demonstrate that CCR1 expression on donor cells contributes to the development of both GVHD and GVL, and suggest that CCR1/CCL5 receptor-ligand interactions modulate allo-specific T-cell responses occurring in this context.

Granulomas are sequestration responses observed in a wide variety of clinical conditions, including mycobacterial infection. We previously reported impaired adaptive, Th1 cell-mediated pulmonary granuloma formation in response to bead-immobilized Mycobacterium bovis-purified protein derivative in aged mice. To reveal determinants of age-related immune deficits, the present study examined the effect of aging on early innate stage pulmonary granuloma formation. Aged mice formed more neutrophil-rich innate granulomas with augmented CXCL2 expression followed by a pattern of rapid decay of tumor necrosis factor-alpha, interleukin (IL)-6, CCL3, and CXCL2. This was associated with enhanced IL-10 expression. Blockade of IL-10 signaling with anti-IL-10 receptor antibody reversed the age-related decay. Intracellular flow cytometric analysis revealed that CD11b(+)Gr-1(+/-) mononuclear phagocytes were the primary leukocyte sources of IL-10 in lungs, and their numbers were increased in aged mice. When exposed to purified protein derivative in vitro, young and old CD11b(+)Gr-1(+/-) mononuclear phagocytes from blood or lung had comparable IL-10 expression, suggesting in vivo signals in the aged environment enhanced the number of IL-10-producing cells in the aged lung. Our findings reveal a novel mechanism of age-associated IL-10 mediated pulmonary immune suppression with the potential to alter downstream adaptive immunity.

Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with a cellular inflammatory response. CD8(+) T cells are implicated in COPD pathogenesis, and their numbers significantly correlate with the degree of airflow limitation. Dendritic cells (DCs) are important sentinel immune cells, but little is known about their role in initiating and maintaining the CD8 T-cell response in COPD. To investigate the mechanisms for CD8(+) T-cell recruitment to the lung, we used resected human lung tissue to analyze chemokine receptor expression by CD8(+) T cells and chemokine production by CD1a(+) DCs. Among 11 surveyed chemokine receptors, only CC chemokine receptor (CCR5), CXC chemokine receptor (CXCR) 3, and CXCR6 correlated with COPD severity as defined by criteria from the Global Initiative for Chronic Obstructive Lung Disease. The CD8(+) T cells displayed a Tc1, CD45RA(+) effector memory phenotype. CD1a(+) DCs produced the respective ligands for CCR5 and CXCR3, CCL3 and CXCL9, and levels correlated with disease severity. CD1a(+) DCs also constitutively expressed the CXCR6 ligand, CXCL16. In conclusion, we have identified major chemokine elements that potentially mediate CD8(+) T-cell infiltration during COPD progression and demonstrated that CD1a(+) mucosal-associated DCs may sustain CD8(+) T-cell recruitment/retention. Chemokine targeting may prove to be a viable treatment approach.

CXCR4 is a major receptor for CXCL12 and is known to participate in multiple physiological systems. The present study tested a second generation CXCR4 antagonist, AMD3465, for effects on highly defined models of Th1- and Th2-cell-mediated hypersensitivity-type pulmonary granuloma formation. Type-1 and type-2 granulomas were induced, respectively, by intravenous challenge of sensitized CBA/J mice with Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg antigen-coated beads. Before challenge, mice were implanted with osmotic pumps releasing AMD3465 at 5 microg/hour (6 mg/kg/day). Compared to vehicle, AMD3465 had minimal effect on type-1 inflammation or cytokine responses in draining lymph nodes, but the type-2 inflammation was significantly abrogated with reductions in lesion size and eosinophil content as well as abrogated interleukin (IL)-5, IL-10, and IL-13 cytokine production in draining lymph nodes. The biased effect of AMD3465 correlated with greater CXCR4 ligand expression in the type-2 model. Treatment during a primary response impaired lymph node IL-2 production after both Mycobacteria bovis purified protein derivative and Schistosoma mansoni egg antigen challenge indicating an unbiased effect during immune induction. In summary, CXCR4 blockade inhibited eosinophil recruitment during type-2 granuloma formation and interfered with primary and secondary T-cell activation events in lymphoid tissue, suggesting potential therapeutic application for chronic hypersensitivity diseases.

The contribution of inducible costimulatory molecule (ICOS) to Th1 and Th2 cell-mediated immune responses was examined in well-defined pathogen antigen-elicited models of cell-mediated granuloma formation. Th1 and Th2 granulomas were respectively induced by intravenous challenge of CBA/J mice with Mycobacteria bovis purified protein derivative (PPD) or Schistosoma mansoni egg (SEA) antigen-coated beads. Effects of anti-ICOS blocking antibody on granulomas and lymphoid responses were assessed during elicitation and sensitization. Anti-ICOS treatment during the elicitation abrogated Th1- but not Th2-cell-mediated granuloma formation. Treatment during sensitization augmented SEA-bead granulomas and Th2 cytokines in lymphoid tissue. Anti-ICOS reduced the primary inflammatory response to PPD- but not to SEA-beads, despite comparable induction of ICOS-ligand and ICOS+ T cells. Treatment did not prevent early development of IFNgamma producing cells. Thus, post-activation effector Th1 activity was subject to ICOS blockade and chronic treatment caused diversion to Th2 dominance likely by eroding Th1 effector function or survival.

Dendritic cell (DC) recruitment is a hallmark event in antigen (Ag)-challenged lungs. We previously reported models for analyzing DC migration and activation in the lung after Th1- or Th2-eliciting pathogen Ag-bead challenge. To determine the role of chemokines in DC mobilization, we applied this analysis to CCR1, CCR2, CCR5, and CCR6 chemokine receptor knockout mice. Both Mycobacteria bovis protein Ags and helminthic, Schistosoma mansoni egg Ags elicited multiple chemokines, including CCR1, CCR2, CCR5, and to a lesser extent CCR6 ligands. DCs from wild-type lungs expressed transcripts for chemokine receptors, CCR1, CCR2, CCR5, and CXCR4. In all knockout strains, CD11c+ cells were recruited to Ag-beads likely because of receptor redundancy. However, DCs in CCR2-/- mice had significantly decreased MHCII and CD40 expression. This was associated with abrogated cytokine production in draining lymph node cultures. Analysis of local innate inflammation revealed a 50% reduction in macrophage recruitment in CCR2-/- mice. Bone marrow chimeras of mixed CCR2+/+ green fluorescent protein transgenic and CCR2-/- green fluorescent protein-negative cells confirmed the DC maturation defect was only among the latter population. In conclusion, CCR2 knockout confers an intrinsic DC activation defect and CCR2 ligands likely promote the local activation/maturation of inflammatory DCs.

Granulomas are innate sequestration responses that can be modified by superimposed acquired immune mechanisms. The present study examined the innate stage of pulmonary granuloma responses to bead-immobilized Th1- and Th2-inducing pathogen antigens (Ags), Mycobacteria bovis purified protein derivative (PPD) and Schistosoma mansoni soluble egg Ags (SEA). Compared to a nonpathogen Ag, PPD and SEA bead elicited larger lesions with the former showing accelerated inflammation. Temporal analyses of cytokine and chemokine transcripts showed all Ag beads induced tumor necrosis factor-alpha mRNA but indicated biased interleukin (IL)-1, IL-6, and IL-12 expression with PPD challenge. All beads elicited comparable levels of CXCL9, CXL10, CCL2, CCL17, and CCL22 mRNA, but PPD beads caused biased CXCL2 CXCL5, CCL3, and CCL4 expression whereas both pathogen Ags induced CCL7. Immunohistochemical, electron microscopic, and flow cytometric analyses showed that Ag beads mobilized CD11c+ dendritic cells (DCs) of comparable maturation. Transfer of DCs from PPD Ag-challenged lungs conferred a Th1 anamnestic cytokine response in recipients. Surprisingly, transfer of DCs from the helminth SEA-challenged lungs did not confer the expected Th2 response, but instead rendered recipients incapable of Ag-elicited IL-4 production. These results provide in vivo evidence that lung DCs recruited under inflammatory conditions favor Th1 responses and alternative mechanisms are required for Th2 commitment.

Monocyte chemotactic protein-3 (MCP-3/CCL7) has potent eosinophil chemoattractant properties. The present study determined its relative contribution to the formation of Th2 cytokine-mediated (type-2) eosinophil-rich interstitial lung granulomas induced by antigens of Schistosoma mansoni eggs. Both MCP-3 transcripts and protein levels were more strongly expressed in lungs with type-2 than with type-1 (mycobacterial antigen-elicited Th1-mediated) granulomas. In vivo treatment with neutralizing antibodies demonstrated that MCP-3 abrogated eosinophil accumulation in type-2 lesions by 40 to 50%. Immunohistochemical staining revealed that MCP-3 localized to vessels in or near granulomas suggesting that endothelial cells were an important in situ source of MCP-3. Maximal MCP-3 transcript expression was abrogated by anti-interleukin-4 treatment. Furthermore, cultured mouse lung endothelial cells displayed augmented MCP-3 production in response to interleukin-4. Together, these results suggest that MCP-3 contributes to a significant component of eosinophil recruitment in the type-2 interstitial granuloma formation and Th2 cytokines promote its production.

Transgenic technology provides one approach for examining cytokine properties in vivo. This study directly tested the effect of a lung-targeted IL-13 transgene on the induction and elicitation of Th1 and Th2 cell-mediated immuno-inflammatory responses. Induction of Th1 (type 1) and Th2 (type 2) responses were tested by sensitization of IL-13 transgenics and littermates with purified protein derivative (PPD) of Mycobacterium bovis or Schistosoma mansoni eggs. Secondary elicitation of pulmonary granulomas was examined in adoptively sensitized transgenics and littermates challenged with bead-bound PPD or S. mansoni egg antigens. Parameters included lymphoid tissue cytokine profiles and granuloma sizes. Results showed that induction and elicitation of both type 1 and type 2 cytokines and granulomas were significantly abrogated in transgenics. Systemic effects were possible, as transgenic serum contained high levels of circulating IL-13. These findings support the concept that IL-13 impairs effector functions and provide novel information regarding its role in regulating Th2 cytokines.

The pathogenesis of end-stage, chronic lung disease is thought to be characterized by an initial inflammatory response followed by fibroproliferation and deposition of extracellular matrix. Many of these chronic lung disorders share a variety of common properties, including an unknown etiology, undefined mechanisms of initiation and maintenance, and progressive fibrosis. Unfortunately, efficacious therapeutic options are not readily available for the treatment of many chronic lung diseases, which may reflect the limited scientific and mechanistic understanding of these disorders. However, recent studies have shown that cytokine networks are likely operative in dictating the progression of these diseases, as these mediators can influence fibroblast activation, proliferation, and collagen deposition during the maintenance of chronic fibrotic lung disease. Accumulating data support the concept that the specific cytokine phenotype may provide a fundamental mechanism for the regulation or continuation of the fibrotic process. For example, interferon-gamma appears to suppresses fibroblast activities, such as proliferation and collagen production, while interleukin (IL)-4 and IL-13 can augment fibroblast growth and collagen production. Interestingly, these mediators are prototypic cytokines that functionally define either a type-1 or a type-2 immune response. Thus, experimental models of cell-mediated lung inflammation, which are characterized by either a type-1 or a type-2 response, will be useful in delineating the mechanisms that either maintain or resolve chronic lung inflammation and accompanying fibrosis.

Transcript expression of 24 chemokines (CKs) was examined throughout 8 days in mouse lungs with type-1 (Th1) or type-2 (Th2) cytokine-mediated granulomas induced by bead-immobilized mycobacterial purified protein derivative or Schistosoma mansoni egg antigens. Where possible, CK protein levels were also measured. In addition, we examined effects of in vivo cytokine depletions. Findings were as follows: 1) bead challenge induced increases in 18 of 24 CK transcripts with type-1 and type-2 responses displaying different patterns. CKs fell into four categories: a) type-1-dominant (gamma-interferon-inducible protein (IP-10), monokine induced by INF-gamma (MIG), macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced chemokine (LIX), rodent growth-related oncogene homologue (KP), macrophage inflammatory protein-1alpha (MIP-1alpha) and -1beta (MIP-1beta), lymphotactin), b) type-2-dominant (eotaxin, monocyte chemotactic protein-2 (MCP-2) and -3 (MCP-3), liver and activation-regulated chemokine (LARC), T cell activation protein-3 (TCA-3), c) type-1 and type-2 co-dominant (MCP-1, MCP-5, monocyte-derived chemokine (MDC), thymus and activation-related chemokine (TARC), C10), and d) constitutive (lungkine, secondary lymphoid-tissue chemokine (SLC), EBI1-ligand chemokine (ELC), fractalkine, macrophage inflammatory protein-1gamma (MIP1-gamma), and stromal cell derived factor-1alpha (SDF1-alpha). 2) CKs displayed characteristic temporal patterns. CXC (IP-10, MIG, MIP-2, LIX, KC) and certain CC (MCP-1, MCP-5, MIP-1alpha, MIP-1beta) CKs were produced maximally within 1 to 2 days. Others (MCP-2, MCP-3, eotaxin, lymphotactin, LARC, TCA-3) displayed peak expression later. 3) Interferon-gamma neutralization profoundly abrogated MIG, but had little effect on other CKs. Tumor necrosis factor-alpha neutralization caused up to 50% reduction in a range of CKs. These findings indicate that type-1 and type-2 granulomas display characteristic CK profiles with coordinated expression that is under cytokine-mediated regulation.

Mice with targeted mutation of chemokine receptor 1 (CCR1) were used to assess the contribution of CCR1 agonists to local, regional, and systemic inflammatory-related events during experimental pulmonary granuloma formation. Models of Th1 (type-1) and Th2 (type-2) cell-mediated lung granulomas were induced in wild-type (CCR+/+) and knockout (CCR1-/-) mice by embolizing Sepharose beads coupled to the purified protein derivative of Mycobacterium bovis or soluble antigens derived from Schistosoma mansoni eggs. Morphometric analysis indicated that granuloma sizes were unchanged in CCR1-/- mice, but flow cytometric analyses of dispersed granulomas revealed that natural killer cell recruitment to type-1 lesions was abrogated by 60%. Analysis of cytokine production by draining lymph node cultures showed altered expression in CCR1-/- mice characterized by reduced interleukin-2 and interferon-gamma in the type-1 response, and enhanced interleukin-5 and interleukin-13 in the type-2 response. Peripheral blood leukocytosis was also enhanced in the type-1 but not the type-2 response. These findings suggest that CCR1 agonists contribute to multiple immunoinflammatory events in the type-1 granulomatous response with natural killer cell accumulation being particularly sensitive to CCR1 disruption. Although functional efficacy of granulomas may be altered, chemokine redundancy and cytokine reserve seem to make the bulk of the exudative response resistant to CCR1 disruption.

Monocyte chemotactic protein (MCP)-1 is postulated to play a role in cellular recruitment during inflammatory reactions. C-C chemokine receptor 2 (CCR2) is considered the major G-protein coupled receptor for MCP-1/JE. We reported that mice with knockout of the CCR2 gene display partially impaired type-1 granuloma formation. The present study similarly examined the effect of CCR2 deficiency on synchronously developing type-2 (Th2) cytokine-mediated lung granulomas elicited by embolization of beads coated with Ags of Schistosoma mansoni eggs. Systemically, blood monocytes were reduced by about half throughout the 8-day study period. At the local level, granuloma size and macrophage content were impaired during the early growth phase (days 1 to 2). By day 4, granuloma sizes were similar to controls. In granulomatous lungs, CCR2 knockout increased mRNA for CCR2 agonists, MCP-1, MCP-3, and MCP-5, but reduced IL-4 and IFNgamma mRNA. The latter was possibly related to decreased CD4+ T cell recruitment. Regionally, draining lymph nodes showed panlymphoid hyperplasia with impaired production of IFNgamma, IL-2, and IL-4, but not IL-5, IL-10, or IL-13. Analysis of procollagen gene expression indicated transient impairment of procollagen III transcripts on day 4 of granuloma formation. These findings indicate that agonists of CCR2 contribute to multiple facets of type-2 hypersensitivity granulomatous inflammation.

Background: Regardless of its involvement in either wound healing or excessive fibrosis, the interstitial fibroblast can now be considered an important early participant in inflammatory responses. Although it is recognized that certain immune cells and proinflammatory mediators are intricately linked to fibrotic disease, little is presently known about the manner in which these mediators and cells are orchestrated to a fibrotic finale. Experimental studies have shown that interstitial fibroblasts are capable of participating in an inflammatory response by promoting direct fibroblast-to-immune cell communication and/or modulating the release of soluble mediators that are mutually recognized by both types of cells., Methods: Primary cultures of murine fibroblasts were recovered from either normal tissue or tissue undergoing a cell-mediated inflammatory response. These stromal cells were assessed for the expression of various cytokines and chemokines indicative of a type 1 or type 2 response. In addition, the fibroblasts were co-cultured with mononuclear cells to assess the cell-to-cell communication., Results: Fibroblasts recovered from different cell-mediated inflammatory responses demonstrated a dramatic alteration in their cytokine profile. Fibroblasts recovered from the type 2 immune response produced high levels of monocyte chemotactic protein-1 (MCP-1), as compared to the normal fibroblasts and fibroblasts recovered from the type 1 lesion. Mononuclear cells co-cultured with fibroblasts induced a contact-dependent expression of elevated levels of chemokines, especially the macrophage-derived MIP-1 alpha. Thus, both fibroblasts themselves and fibroblasts co-cultured with immune-inflammatory cells have the ability to participate in the maintenance of an inflammatory response via the expression of chemokines., Conclusions: Our laboratory and others have addressed the role of chemotactic cytokines or chemokines in the fibrotic process, and have demonstrated that fibroblasts are capable of modulating the activation of various immune cells that have been implicated in fibrotic disease. In addition, the interstitial fibroblast is capable of regulating its own behavior within the interstitial environment via the expression of chemokines and chemokine receptors. Thus, novel strategies aimed at preventing fibrotic disease will likely need to address the early engagement of inflammatory cells by fibroblasts, and possibly modulate the ability of fibroblasts to generate and/or recognize profibrotic signals supplied by chemokines.

Recent studies support the concept that pulmonary granulomatous inflammation directed by interferon (IFN)-gamma, interleukin (IL)-12, and nitric oxide usually resolves in the absence of fibrosis. To determine whether nitric oxide participates in modulating the fibrotic response during the development of pulmonary granulomas in response to purified protein derivative (PPD), mice presensitized to PPD received daily intraperitoneal injections of N(G)-nitro-D-arginine-methyl ester (D-NAME), N(G)-nitro-L-arginine-methyl ester (L-NAME), or aminoguanidine after delivery of PPD-coated beads to the lungs. Eight days later, morphometric analysis of lung granulomas revealed that L-NAME-treated mice when challenged with PPD in vitro for 36 hours had the largest pulmonary granulomas and the greatest collagen deposition among the treated groups. In addition, equivalent numbers of dispersed lung cells from L-NAME- and aminoguanidine-treated mice produced significantly higher levels of IL-4, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1alpha and significantly lower levels of eotaxin compared with D-NAME-treated mice. Cultures of dispersed lung cells from L-NAME-treated mice also produced significantly more IL-10 and less IL-12 compared with similar numbers of dispersed lung cells from D-NAME-treated mice. Cultures of isolated lung fibroblasts from L-NAME-treated mice expressed higher levels of C-C chemokine receptor 2 (CCR2) and CCR3 mRNA and contained less MCP-1 and eotaxin protein than a similar number of fibroblasts from D-NAME-treated mice. Thus, nitric oxide appears to regulate the deposition of extracellular matrix in lung granulomas through the modulation of the cytokine and chemokine profile of these lesions. Alterations in the cytokine, chemokine, and procollagen profile of this lesion may be a direct effect of nitric oxide on the pulmonary fibroblast and provide an important signal for regulating fibroblast activity during the evolution of chronic lung disease.

The expression of cytokines can dictate the intensity, chronicity, and type of immune/inflammatory response that is produced. These events may be regulated by accumulation of particular cell populations at a site of immune response that can be regulated by the expression of specific chemokines. Recent data have indicated that chemokines also have direct effects on cellular activation. In particular, T lymphocyte responses have been divided into two distinct phenotypes, designated by TH1- and TH2-type cytokine expression. Although it is recognized that divergent T-lymphocyte-derived cytokine phenotypes exist, the mechanisms that dictate the expression of these cytokines and ultimately the division of these immune responses is not entirely clear. In the present study, we present data that the C-C chemokine family members may be a factor influencing the direction of T-cell-derived lymphokine production. To elucidate the role of C-C chemokines, MIP-1 alpha and MCP-1, we have used both antigen-specific (schistosomal egg antigen (SEA)) and nonspecific (conconavalin (Con) A) stimuli. Using TH2-type lymphocyte populations from SEA-sensitized mice, a significant increase in IL-4 mRNA expression and protein production was observed when MCP-1 was added to the culture. Conversely, MIP-1 alpha treatment appeared to decrease interleukin (IL)-4 production. Interestingly, the proliferative response in the TH2-type (SEA-specific) response was up-regulated by MIP-1 alpha whereas MCP-1 down-regulated the response, inversely correlating with IL-4 production. Primary stimulation of naive lymphocytes with Con A induces a predominant interferon (IFN)-gamma response, whereas the second stimulation of the same lymphocytes with Con A induces both IFN-gamma and IL-4. When the two C-C chemokines were individually co-incubated with Con-A-stimulated lymphocytes, both up-regulated IFN-gamma production and proliferation during the primary stimulation. Similarly, in the secondary response, both chemokines further upregulated IFN-gamma production; however, only MCP-1 co-stimulation increased IL-4 production, whereas MIP-1 alpha significantly decreased IL-4 production in these same cell populations. These results were also reflected in steady-state levels of mRNA expression. These results suggest that the production of C-C chemokines (MCP-1 or MIP-1 alpha) during an immune response may aid in determining the type of cytokines produced and the level of lymphocyte activation during a particular response.

The present study explored the role of murine monocyte chemotactic protein (MCP) in the T cell-mediated hypersensitive granulomatous response to Schistosoma mansoni eggs. The study examined the time course of local production, contribution to cellular infiltration, and the role of T cells in endogenous regulation. Synchronized pulmonary granulomas were induced under conditions of primary and secondary states of immunity. Primer-directed polymerase chain reaction analysis showed increased MCP mRNA expression in granulomatous lungs, mainly in the secondary response. Levels of MCP were measured by enzyme-linked immunosorbent assay in cultures of intact granulomas. Spontaneous MCP production was modest in primary granuloma cultures, reaching a maximum of 5.7 +/- 0.9 ng/ml by 16 days. In contrast, the secondary response showed augmented and accelerated production, achieving 13 +/- 2.0 ng/ml by 2 days. Immunohistochemical staining revealed the strongest MCP expression within microvascular adventitial cells or pericytes as well as in scattered mononuclear cells associated with granulomas. Staining was not detected in normal lungs. Passive immunization with anti-MCP-1 antibodies caused a 40% reduction in the secondary granuloma area but did not significantly affect the primary response. With adoptive cell transfer and T cell subset depletion, it was shown that Thy-1+ and CD5+ cells augmented, whereas CD8+ cells appeared to impair, MCP production. This provides direct evidence that MCP is involved in secondary Th2-mediated response to schistosome eggs and is subject to regulation by T cells.

Synchronized pulmonary granulomas (GRs) were induced in presensitized mice by intravenous embolization of polymer beads bound with purified protein derivative (PPD) of Mycobacteria tuberculosis or soluble antigens derived from Schistosoma mansoni eggs (SEA). Uncoated beads served as a foreign body control (CON). Antigen-coated beads elicited GRs with characteristic epithelioid macrophages and multinucleate giant cells by 4 days after embolization. Unlike PPD GR, SEA bead lesions contained eosinophils, whereas CON beads elicited only a limited mononuclear infiltrate. GRs and draining lymph nodes (LN) were assessed on days 2, 4, and 8 for Th1-(interleukin-2 [IL-2], interferon-gamma[IFN] and Th2-type (IL-4, IL-5, and IL-10) cytokines. CON GR produced only a small amount of IFN-gamma on day 2 and failed to induce a significant response in draining LN. In contrast, both PPD and SEA antigen-coated beads induced reactive lymphoid hyperplasia but differed greatly in local and regional cytokine profiles. PPD GR produced IFN-gamma on day 2 and the draining LN produced predominantly Th1 cytokines on days 2 and 4. In contrast, SEA beads GRs were dominated by Th2 cytokines. The corresponding LN produced IL-2 and IL-4 on day 2; IL-2, IL-4, IFN-gamma, and IL-10 on day 4; then IL-2, IFN-gamma, and IL-4 on day 8, probably reflecting maturational changes of T cells. Macrophages (MP) from bead GR also showed different patterns of IL-6 and tumor necrosis factor (TNF) production. Compared with CON GR, MPs from PPD GR were weak sources of IL-6, whereas those of SEA GR showed enhanced and accelerated production. In contrast, MP of PPD GR had augmented TNF-producing capacity, whereas those of SEA GR showed delayed TNF production. In vivo depletion of TNF, respectively, caused 40 and 10% decreases in PPD GR and SEA GR but had no effect on CON GR area, indicating that TNF contributed to a greater degree to the PPD response. These data show that depending on the inciting agent, GR can be mediated by different cytokines. Characterization of inflammatory lesions by cytokine profiles should allow design of more rational therapeutic interventions.

The formation of hepatic granulomas around persistently deposited Schistosoma mansoni eggs leads to parenchymal damage, ongoing fibrosis, and ultimate loss of liver function. In this study, the production of macrophage inflammatory protein-1 alpha (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) by granuloma fibroblasts was examined to establish the potential contribution of intragranuloma fibroblasts to the maintenance of the chronic inflammation. Isolated fibroblasts from dispersed acute infection hepatic granulomas were grown in tissue culture for 3 to 4 weeks and used on the third or fourth passage. We initially surveyed fibroblasts for production of MIP-1 and MCP-1 by reverse transcription-polymerase chain reaction (RT-PCR) after stimulation with interleukin (IL)-1, tumor necrosis factor, interferon (IFN)-gamma, IL-4, or IL-10: cytokines found within the granuloma. These studies demonstrated constitutive expression of MCP-1 and differential up-regulation of MIP-1 on cytokine stimulation. Protein expression was then verified by immunohistochemical localization of MIP-1 and MCP-1 in paraformaldehyde-fixed fibroblasts and by direct quantitation of MIP-1 and MCP-1 in culture supernatants by specific ELISAs. These studies demonstrated constitutive expression of MCP-1 in unstimulated and cytokine-stimulated granuloma fibroblasts. In contrast, IL-1 (0.1 to 2.5 ng/ml), IFN-gamma (10 micrograms/ml), and IL-10 (2.5 to 10 ng/ml) were able to induce the significant production of MIP-1 by the granuloma fibroblasts. Interestingly, normal noninflammatory fibroblasts from uninfected mice showed no significant production of MIP-1 or MCP-1 in response to these cytokines. These results suggest that granuloma fibroblasts may be phenotypically altered compared with normal fibroblasts and have a significant role in leukocyte recruitment, granuloma growth, and maintenance of the egg-induced lesion.

Bronchogenic carcinoma displays an aggressive clinical course that may reflect a capacity to evade host defenses. We postulated that tumors may elaborate interleukin-1 receptor antagonist protein (IRAP) to escape host interleukin-1-dependent responses. Homogenates of human bronchogenic lung tumors demonstrated significant increases of IRAP compared with normal lung tissue controls (n = 48). There was no significant difference in interleukin-1 beta levels between tumor and normal lung tissue. Immunohistochemical staining localized IRAP to tumor cells. Semiquantitative pathological analysis demonstrated a modest inflammatory cell infiltrate with qualitative differences between tumors of different histology. Western blot analysis of tumor homogenates demonstrated several molecular weight forms of IRAP. Finally, antigenic IRAP was detected in supernatants of the human bronchogenic carcinoma cell line (A549) maintained in vitro. These findings illustrate the capacity of bronchogenic tumors to produce and secrete IRAP that may be important in tumor evasion of host defenses.

During acute inflammation, the first line of cellular response for host defense is the neutrophil. In addition to the historic role of the neutrophil as a phagocyte, recent studies have identified this cell as an important source of a number of cytokines. In this study, we provide evidence that the neutrophil is a significant source of interleukin-8 (IL-8). Neutrophils freshly isolated from whole blood were not found to constitutively express IL-8 mRNA. In contrast, when these leukocytes were cultured on plastic they were activated, leading to the significant expression of de novo steady-state levels of IL-8 mRNA. In addition, when neutrophils were treated with cycloheximide, there was evidence for "superinduction" of steady-state levels of IL-8 mRNA and inhibition of antigenic IL-8 production. Neutrophils were subsequently stimulated with lipopolysaccharide (LPS), tumor necrosis factor-alpha, or interleukin-1-beta and were found to express IL-8 mRNA and antigen in both a time- and dose-dependent manner. Furthermore, neutrophils stimulated with traditional chemotactic/activating factors, such as the split product of the fifth component of complement (C5a), formylmethionyleucylphenylalanine (fMLP), and leukotriene B4 (LTB4) in a dose-dependent manner did not produce significant antigenic IL-8, as compared with unstimulated controls. In contrast, when neutrophils were exposed to either of these neutrophil agonists in the presence of LPS, the production of antigenic IL-8 was significantly elevated, as compared with either of the stimuli alone, suggesting a synergistic response. These data would suggest that the neutrophil can no longer be viewed as only a phagocyte or warehouse for proteolytic enzymes, but is a pivotal effector cell that is able to respond to mediators in its environment and generate cytokines. This latter neutrophil response may be important for either the elicitation of additional neutrophils or to orchestrate the conventional immune response at sites of inflammation.

Interleukin-6 (IL-6) is considered an important multifunctional cytokine involved in the regulation of a variety of cellular responses, including the induction of acute-phase protein synthesis, lymphocyte activation, and hematopoiesis. In vitro studies have identified many cells that can produce IL-6, but the cellular sources under physiologic conditions have yet to be identified. Using immunoaffinity purified goat anti-murine IL-6, the authors performed immunohistochemical studies to localize cells expressing IL-6 in selected organs of normal and endotoxin challenged NIH-Swiss outbred mice. In the blood, findings were correlated with cell-associated bioactivity using the standard B9 cell proliferation assay. In normal mice, constitutive expression was seen in granulocytes, monocytes and their precursors as well as in bone marrow and splenic stromal macrophages. Hepatic macrophages were negative, as were lymphocytes, megakaryocytes, erythroid precursors, and endothelial cells. In the absence of significant serum levels of IL-6, cell-associated IL-6 bioactivity was detected in circulating polymorphonuclear leukocytes (PMNs), but not lymphocytes. After endotoxin challenge, there was a threefold increase in PMN IL-6 content from 1 to 3 hours followed by almost complete depletion at 6 hours. This correlated well with a threefold increase of IL-6 mRNA in the bone marrow followed by a decrease at 6 hours. This pattern also correlated with serum levels of IL-6, which peaked at 3 hours and dropped significantly by 6 hours. By 24 hours, cell-associated IL-6 showed recovery with no increase in serum levels. In vivo findings showing IL-6 expression in bone marrow macrophages support in vitro studies suggesting a role for IL-6 in hematopoiesis. Furthermore, PMNs as well as macrophages are likely important sources of IL-6 during inflammatory and septic states.

Tumor necrosis factor (TNF)-alpha has significant biologic actions in many circumstances, such as infectious diseases, ischemia/reperfusion injury, and delayed-type hypersensitivity reactions. Based on the hypothesis that manipulation of TNF can play an important role in treatment of heart transplant rejection, the objective of this study was to determine whether anti-TNF antibodies could prolong cardiac allograft survival. Hearts from brown rats were transplanted to the necks of recipient Lewis rats. Graft survival was determined by direct palpation of the heart; complete graft rejection was defined by cessation of contraction. In untreated rats, the hearts were rejected 6.8 +/- 0.6 days (n = 10; mean +/- SEM) after transplantation. The mononuclear cell infiltrate in the transplanted hearts stained intensely for TNF by immunohistochemistry, indicating that TNF was present within the inflammatory cells associated with the rejection process. In rats receiving a single injection of anti-TNF antibody at the time of transplantation (n = 6), however, graft survival was nearly doubled (12.7 +/- 1.4 days; p less than 0.001 vs controls). Prolonged cardiac graft survival was also evident if the anti-TNF treatment was delayed until 1 day (n = 5; rejection at 16.2 +/- 2.4 days; p less than 0.001 vs controls) or even 3 days after transplantation (n = 5; rejection at 11.4 +/- 2.3 days; p less than 0.005 vs controls). Treatment at 5 days after transplantation, however, was not effective (n = 3; rejection at 7.7 +/- 0.6 days; p, not significant vs controls). The data indicate that a single bolus of anti-TNF antibodies can delay heart transplant rejection, even when administered up to 3 days after implantation, supporting the potential utility of anti-TNF therapy for treatment of heart transplant rejection.

Human interleukin 1 receptor antagonist protein (IRAP) is a specific antagonist of interleukin 1 (IL-1) action in both in vitro and in vivo experimental models. Presently, the significance of this protein in human pathophysiology is unknown. In the present study, monoclonal antibodies against IRAP were prepared and used to demonstrate IRAP expression in human tissues, immunohistochemically. Specifically, this study focused on lymphoid tissues and granulomatous inflammatory reactions since IL-1 is believed to play roles in lymphocyte development and inflammation. In addition, these tissues were also stained for IL-1 beta to compare the expression of agonist and antagonist. These findings indicate that IRAP expression is largely limited to macrophages and their derivatives. Strong IRAP expression was observed in germinal center macrophages of lymph nodes, spleen, and tonsil. In contrast, IL-1 was marginally expressed in these organs. Granulomas associated with active M. tuberculosis infection, sarcoidosis and foreign bodies all contained strongly IRAP positive cells, which included macrophages, epithelioid cells and multinucleate giant cells. Unlike reactive lymphoid tissue, tuberculous and sarcoid granulomas also contained IL-1 positive cells which included macrophages and their derivatives, as well as some stromal cells. Foreign body lesions showed minimal IL-1 expression. Interestingly, granulomas in patients with acquired immunodeficiency disease (AIDS) associated with M. avium-intracellulare contained IRAP positive cells but were negative for IL-1 expression. Taken together, these findings suggest that IRAP takes part in both physiologic and pathologic reactions. Moreover, they provide a basis to design future studies to determine the precise contribution of IRAP to these reactions.

The signals resulting in leukocytes infiltrating the tubulointerstitial compartment during renal inflammatory disease are not well understood. A recently described cytokine, interleukin-8 (IL-8), has been demonstrated to be chemotactic for lymphocytes and neutrophils at picomolar and nanomolar concentrations, respectively. Cytokeratin positive, renal cortical epithelial cells (RCEC) with tubular attributes were cultured from kidney tissue from six human subjects. We report that these human renal cortical epithelial cells in primary cell culture respond to either IL-1 beta, TNF or LPS in both a time- and dose-dependent manner by expressing IL-8 mRNA and secreting antigenic IL-8 peptide. In addition, RCEC were found to be strongly positive for cell-associated antigenic IL-8 peptide by immunostaining after 24 hour incubation with IL-1 beta, TNF and LPS. To ascertain whether IL-8 was present in renal disease associated with infiltrating leukocytes, we performed immunohistochemistry on renal biopsy specimens from patients with acute allograft rejection. Both proximal and distal tubular epithelial cells were found to be strongly positive for cell-associated antigenic IL-8. These findings suggest that the human renal tubule epithelial cell may actively participate in acute inflammatory processes in the kidney, including allograft rejection, by effecting and directing leukocyte chemotaxis via the production of IL-8.

Human mesangial cells (MC) in culture, when stimulated by interleukin 1 alpha(IL-1 alpha) or tumour necrosis factor (TNF alpha), but not with lipopolysaccharide (LPS), express interleukin 8 (IL-8) mRNA, and both cell associated and extracellular IL-8. Dexamethasone treatment of mesangial cells induced partial inhibition of the release of extracellular IL-8, while cell-associated IL-8 and IL-8 mRNA were not significantly altered. We propose that the mesangial cell has a direct role in the initiation and propagation of inflammatory events within the glomerulus via the generation of the chemotactic cytokine IL-8.

Using a model of sepsis induced by parenteral challenge of mice with bacterial lipopolysaccharide (LPS), the authors analyzed the in vivo expression of interleukin-1 (IL-1) alpha,beta and tumor necrosis factor (TNF). Both TNF and IL-1 alpha,beta were detected in hepatic sinusoidal macrophages (Kupffer cells), immunohistochemically. Kinetic analysis showed a clear sequence of synthesis. Tumor necrosis factor was produced first, reaching maximal expression at 1 hour after LPS challenge, then rapidly disappeared. IL-1 beta followed, reaching maximal expression at 2 to 3 hours, then dropped off by 6 hours. Interleukin-1 alpha expression reached a peak at 6 hours and had disappeared by 18 hours. Analysis of serum bioactivity also revealed sequential expression that correlated with immunohistochemical findings. Tumor necrosis factor was maximal at 1 hour and IL-1 at 6 hours. The IL-1 bioactivity was not due to interleukin-6 (IL-6), as this was depleted from specimens by immunoabsorption. Also IL-6 bioactivity reached maximal levels at 3 hours, earlier than IL-1. Pretreatment with 4 mg/kg dexamethasone significantly decreased Kupffer cell expression of TNF and IL-1 alpha (about 80% and 60% suppression, respectively) but had less effect on IL-1 beta expression (about 30% suppression). Accordingly, serum levels of TNF were suppressed by 75% while serum IL-1 was decreased by 39%, indicating differential sensitivity of these cytokines to glucocorticoids. Endogenous corticosteroid levels increased as TNF levels decreased, supporting the contention that glucocorticoids regulate TNF synthesis. In contrast, IL-1 levels rose concurrently with corticosterone. These data indicate a sequential activation of cytokine gene expression in vivo, which may be critical to the cascade of events leading to septic shock, and provide evidence that Kupffer cells are a major source of cytokines in endotoxemia. Finally, the differential sensitivity of cytokine expression to glucocorticoids may in part explain the inadequacy of the latter in the treatment of sepsis.

Chemotactic cytokines are becoming increasingly recognized as important participants in the coordinate recruitment of specific inflammatory cells. In this manuscript we present data demonstrating that LPS challenged human mononuclear phagocytic cells can express mRNA for neutrophil chemotactic factor/interleukin-8 (NCF/IL-8), but do not express mRNA for monocyte chemotactic protein (MCP). The expression of NCF/IL-8 mRNA was time and dose dependent. This identical stimulus response was also found in peripheral blood neutrophils. These studies demonstrate a disparate production of chemotactic cytokines by macrophages and exemplify the dynamic nature of the chemotactic response.

Synchronous models of T-cell-mediated and foreign body granulomas were induced in mice by intravenous embolization of Schistosoma mansoni eggs and Sephadex beads, respectively. The authors then performed flow-cytometric analysis of lymphocytes from dispersed granulomas, spleens, and peripheral blood at 4, 8, 16, and 32 days corresponding to the induction, growth, and maintenance, and resolution of these lesions. Lymphocytes were identified on the basis of light scatter characteristics, and the nature of the cells was confirmed by cell sorting and electron-microscopic examination. Lymphocyte subpopulations were characterized with antibodies to lymphocyte surface markers, specifically Ig, Thy 1.2, Lyt 1, Lyt 2, and L3T4. Natural killer cells were identified with anti-asialo GM1. Egg-induced granulomas had more lymphocytes of all phenotypes at all time points. Surprisingly, there was a significant number of cells staining positive for asialo GM1. On Day 16 after embolization there was a greater percentage of helper T cells, as defined by positive staining with L3T4, in the egg model, compared with the bead model. There was no obvious shift of lymphocytes from either the blood or spleen into the granuloma. These data confirm the importance of T cells in the direct participation of granulomatous inflammation, and the large numbers of asialo GM1-positive cells suggest a role for natural killer cells.

A number of cytokines are active during the evolution of an inflammatory response, including tumor necrosis factor-alpha, interleukin-1, and novel chemotactic cytokines. This latter group of mediators belong to supergene families of inflammatory signals that play a key role in the selective recruitment of immune cells. In this presentation, we present data demonstrating, for the first time, endothelial cell expression of monocyte chemotactic protein (MCP) mRNA induced by LPS, interleukin-1 or tumor necrosis factor. Human fibroblasts were also found to express monocyte chemotactic factor mRNA in response to interleukin-1 or tumor necrosis factor, but LPS was not effective. In addition, neither primary cultures expressed MCP in response to interleukin-6. These studies demonstrate that non-immune "bystander" cells can play an active role in the recruitment of inflammatory cells via the production of novel chemotactic cytokines.

The effect of parenteral administration of prostaglandins, 15-(s)-15-methyl PGE1 (M-PGE) and PGF2 alpha (PGF) on the pathophysiologic manifestations of active murine Schistosoma mansoni infection was examined. Both M-PGE and PGF resulted in a nearly 50% suppression of granuloma size following a 7-day course of treatment. M-PGE and PGF appeared to act by different mechanisms. The former caused a broad-spectrum immunosuppression manifested as reduced splenomegaly, B-cell proliferation, and antigen-specific interleukin-2 (IL-2) production as well as decreased granuloma macrophage Ia antigen expression, superoxide anion (O2-) production, and interleukin-1 (IL-1) production. In contrast, PGF did not ameliorate splenomegaly, but caused increases in splenic asialo-GM1 (natural killer cells) and L3T4 (helper) positive T cells. Prostaglandin F also reduced IL-2 production, but to a lesser extent that M-PGE. Although PGF caused reduced granuloma macrophage Ia expression and O2- production, it did not suppress IL-1 production. Overall, these data show that PGs can significantly modulate immunopathologic events in chronic granulomatous disease states.

The role of oxygen reactive species in granulomatous hypersensitivity was explored using a model of pulmonary granulomas induced by intravenous injection of eggs from the parasite Schistosoma mansoni. Macrophages from sychronously developing lesions spontaneously released significant quantities of superoxide anion (26 nmoles/10(6)/2h) by 8 days of development. In contrast, the non-T cell foreign body (Sephadex bead) granuloma macrophages produced only (2 nmoles/10(6)/2h) small quantities. Daily administration of the oxygen scavenger, alpha-tocopherol, by either oral or parenteral routes caused up to 60% suppression of granuloma size. Moreover, parenteral administration of specific inactivators of 0-2 and H2O2, superoxide dismutase and catalase respectively, resulted in a 30 to 40% reduction in granuloma size. These data suggest that oxygen reactive species take part in the generation of hypersensitivity granulomas.

The ability of arachidonic acid (AA) metabolites to regulate I-region-associated (Ia) antigen expression on macrophages from schistosome-egg-induced pulmonary granulomas was examined. The prostaglandin (PG) analog 15-S-15-CH3-PGE1 (M-PGE1) and PGF2 alpha were found to modulate the kinetics of Ia expression when administered in vivo. Methyl-PGE1 significantly suppressed Ia antigen expression by hypersensitivity granuloma macrophages, while PGF2 alpha appeared to potentiate the expression. Lymphokine-induced Ia antigen expression by cultured granuloma macrophages was likewise dramatically inhibited by M-PGE1. Further analysis using systemically administered inhibitors of AA metabolism demonstrated that the cyclooxygenase inhibitor indomethacin caused augmentation of Ia expression. In contrast, lipoxygenase inhibitors significantly reduced both Ia expression and granuloma size. The role of AA metabolites in modulating chronic inflammation is discussed.

Using highly specific polyclonal antisera raised against recombinant murine IL-1 alpha and beta, we performed solid-phase immunoabsorption studies on supernates of resident and adjuvant-elicited CBA/J mouse peritoneal macrophages. Antibody specificity was established by reciprocal absorption studies and Western blot analysis. Supernates obtained from macrophages cultured for 18 hr in the presence of 1 microgram/ml lipopolysaccharide (LPS) were subjected to immunoabsorption. Approximately 78-90% of the released bioactive material was IL-1 and about 80% of this could be attributed to IL-1 beta. Analogous to that reported for human monocytes, these data suggest that IL-1 beta is the predominant released form of IL-1.

We have investigated the effect of tumor necrosis factor on the release of interleukin-1 and PGE2 from murine resident peritoneal macrophages. Tumor necrosis factor causes an increase in the production of interleukin-1 and PGE2 with a maximum induction for both noted at 5.9 X 10(-8) M. While indomethacin decreased tumor necrosis factor induced PGE2 production, this cyclooxygenase inhibitor augmented tumor necrosis factor induced interleukin-1 production. Our data suggests that tumor necrosis factor may be an important immunopotentiating agent in addition to its previously described cytolytic and metabolic activities.

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.

We have investigated the role of arachidonic acid metabolites in the regulation of interleukin-1 production by murine peritoneal macrophages. Indomethacin a potent inhibitor of prostaglandin synthesis caused a dose-dependent augmentation of lipopolysaccharide induced interleukin production (up to 7-fold at 5 microM). In contrast, lipoxygenase inhibitors, nordihydroguarietic acid and nafazatrom had no effect at doses that did not significantly decrease prostaglandin synthesis. Added to lipopolysaccharide stimulated cultures, PGE2 was also augmented by indomethacin but unlike lipopolysaccharide treated cultures was suppressed by nordihydroguarietic acid. These data suggest that arachidonate metabolites may be potent autoregulators of macrophage interleukin-1 production.

The inflammatory modulating activity of specific prostaglandins has been examined for both immune and foreign-body types of pulmonary granulomas. Lung granuloma formation generated in mice by embolization of Schistosoma mansoni eggs was markedly suppressed by treatment with the stable, functional analog of prostaglandin E, (PGE1), (15-(S)-15-methyl PGE1) while treatment with PGF2 alpha augmented the granulomatous response. Despite marked effects on egg-induced granuloma formation, PGs had no significant effect on the foreign body lesion induced by Sephadex beads. Likewise, PGs had no effect on the primary antibody response to schistosome egg antigens. However, notable derangements in splenic lymphoid populations occurred. While T-cell numbers appeared constant in face of PG treatment, B-cell populations were depressed by methyl-PGE1 and augmented by PGF2 alpha. Further analysis revealed that methyl-PGE1 appeared to suppress both the induction and elicitation phases of the cell-mediated response to schistosome eggs. Cyclophosphamide treatment could partially reverse this suppression, but the induction of suppressor cell activity was not solely responsible for this effect. The possible role and mechanism of PGs as modulators of chronic inflammation is discussed.

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.

Using a highly specific rabbit antisera directed against murine tumor necrosis factor (TNF), immunohistochemical localization of this monokine was performed in cultured mouse peritoneal macrophages. Resident macrophages did not express TNF even after stimulus with lipopolysaccharide (LPS). In contrast, 12% of macrophages elicited with Freund's adjuvant stained positively and up to 60% were positive after LPS stimulation. Analysis of the kinetics of expression revealed that maximal staining occurred from 1-3 hours after stimulus with disappearance of staining by 12 hours. Both a membrane and cytoplasmic pattern of staining could be demonstrated. The presence of plasma membrane TNF was confirmed by scanning electron microscopy. Northern blot analysis and bioassay revealed that the kinetics of TNF mRNA synthesis corresponded to the appearance of the protein while its disappearance corresponded to the appearance of TNF in the supernate. Thus, TNF synthesis and secretion could be histochemically demonstrated. These findings support the notion that TNF production is a characteristic of activated macrophages and that such cells display membrane-associated TNF at least transiently after stimulation.